Open pore biodegradable matrices

ABSTRACT

The invention is directed to a process for preparing porous polymer materials by a combination of gas forming and particulate leaching steps. The invention is also directed to porous polymer material prepared by the process, particularly having a characteristic interconnected pore structure, and to methods for using such porous polymer material, particularly for tissue engineering.

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/042,198, filed Mar. 31, 1997.

The invention is directed to a process for preparing porous polymermaterials by a combination of gas foaming and particulate leachingsteps. The invention is also directed to porous polymer materialprepared by the process, particularly having a characteristicinterconnected pore structure, and to methods for using such porouspolymer material, particularly for tissue engineering.

The lack of autologous and allogeneic tissue suitable fortransplantation has driven the development of the tissue engineeringfield, in which new tissues are created from cultured cells andbiomaterials. This is advantageous because these cells can be expandedin vitro and cultured for use by multiple patients. The biomaterialserves as a vehicle to localize the cells of interest, a physical spacerto create potential space for tissue development, and as a templateguiding tissue regeneration. Biodegradable homopolymers and copolymersof lactic and glycolic acid are attractive candidates for fabricatingtissue engineering matrices due to their flexible and well definedphysical properties and relative biocompatability. Additionally, thedegradation product of these polymers are natural metabolites and arereadily removed from the body.

Several techniques have been used to fabricate polymers into porousmatrices for tissue engineering applications, includingsolvent-casting/particulate leaching (SC/PL) (A. G. Mikos, A. J.Thorsen, L. A. Czerwonka, Y. Bao, and R. Langer, “Preparation andcharacterization of poly(L-lactic acid) foams,” Polymer, 35, 1068-1077(1994)); phase separation (H. Lo, M. S. Ponticiello, and K. W. Leong,“Fabrication of controlled release biodegradable foams by phaseseparation,” Tissue Engineering, 1, 15-28 (1995)); fiber extrusion andfabric forming processing (J. F. Cavallaso, P. D. Kemp and K. H. Kraus,“Collagen Fabrics as Biomaterials,” Biotechnology and Bioengineering,43, p. 781-791 (1994)); and gas foaming. (D. J. Mooney, D. F. Baldwin,N. P. Suh, J. P. Vacanti, and R. Langer, “Novel approach to fabricateporous sponges of poly(D,L-lactic-co-glycolic acid) without the use oforganic solvents,” Biomaterials, 17, 1417-1422 (1996).) Thesolvent-casting/particulate leaching and phase separation approachesrequire the use of organic solvents. Residues of organic solvents whichcan remain in these polymers after processing may damage transplantedcells and nearby tissue, and inactivate many biologically active factors(e.g., growth factors) one might wish to incorporate into the polymermatrix for controlled release. Fiber forming typically requires hightemperatures (above the transition temperature of polymer), and is notamenable to processing amorphous polymers. The high temperatures used inthis process would likely denature any biologically active molecules onemight wish to incorporate into the matrix.

The gas foaming method (for example, of Mooney et al., cited above)provides a technique to fabricate highly porous matrices frompoly(lactic-co-glycolic acid) (PLGA) using a high pressure gas thatavoids the use of organic solvents and high temperatures. However, thetechnique typically yields a closed pore structure, which isdisadvantageous in many applications of cell transplantation. Inaddition, a solid skin of polymer results on the exterior surface of thefoamed matrix and this may lead to mass transport limitations.

An object of this invention is to provide a new process for preparingporous polymer materials which are useful for tissue engineering andother applications wherein the pore structure is particularlyadvantageous. For example, the polymers of the invention may have twotypes of porosity, the first formed by gas-foaming processing and thesecond formed by the action of particulate leaching. The combination ofthese two porosity types can be regulated by the processing conditionsand materials used to provide porous polymer materials with a range ofadvantageous properties. In a preferred embodiment, the porosity fromparticulate leaching results in interconnected pore structure materialshaving an open pore structure. Other objects of the invention includethe porous polymer materials prepared by the process and methods usingsuch materials for tissue engineering, for example.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

According to the process of the invention, a mixture of polymerparticles and a leachable particulate material molded, optionally withcompression, to a desired size and shape are subject to a high pressuregas atmosphere so that the gas dissolves in the polymer; then athermodynamic instability is created, for example by reduction of thepressure, so that the dissolved gas nucleates and forms gas pores withinthe polymer; this causes expansion of the polymer particles, and as theyexpand they fuse, creating a continuous polymer matrix containing theparticulate material; finally, the particulate material is leached fromthe polymer with a leaching agent creating a further porosity. Theprocess thus provides a novel combination of the processes of gasfoaming (GF) to form pores and particulate leaching (PL) to form anothertype of porosity. Hence, the process can be termed as a GF/PL process asopposed to the known solvent-casting/particulate leaching (SC/PL)processes.

The novel materials prepared by the process are characterized by havingpores formed from gas foaming and pores formed by particulate leaching,the particulate leaching pores also being termed macropores. Preferably,the porosity resulting from the particulate leaching, which can becontrolled by the amount and size of the particulate among otherfactors, is such that it results in interconnections and, thus, an openpore structure. Typically, matrices prepared by the GF/PL method of theinvention will have an interconnecting or open pore structure akin tothe structure demonstrated in the photomicrographs generated accordingto Example 1 and discussed therein. In one embodiment providing amixture of polymer and leachable particulate wherein the amount ofleachable particulate is at least 50% by volume will result in apartially interconnecting or open pore structure. A higher amount ofleachable particulate can be used to obtain a fully interconnectedstructure.

While materials prepared by an SC/PL process can also provide someextent of an interconnected pore matrix, the inventors have discoveredthat the materials prepared by the inventive GF/PL process exhibit adistinct pore structure and significantly advantageous mechanicalproperties over SC/PL prepared materials. This advantage is in additionto the advantage of the absence of necessity for organic solvents and/orhigh temperatures in preparation of the material and the absence oforganic solvent residue in the prepared materials, which advantages makethe materials even more useful for the applications described below. Forexample, the materials of the invention exhibit much higher strengthproperties, e.g. tensile strength. For instance, the materials accordingto the invention can be prepared to maximize the tensile strength toprovide materials with a tensile modulus of, for example, 850 kPa,particularly 1100 kPa, or higher. Although, such high strength materialsmay not be required for all applications and materials with a tensilemodulus as low as 100 kPa, for example, have been found to be useful.Further, the materials exhibit improved compression resistance. Forinstance, the materials according to the invention can be prepared tomaximize the compression resistance to provide materials with acompression modulus of, for example, 250 kPa, particularly 289 kPa, orhigher. Comparative SC/PL prepared materials exhibit a tensile modulusof about 334±52 kPa and a compression modulus of about 159±130 kPa.

While not intending to be bound by this theory, it is reasonablyhypothesized that the improved mechanical properties and stronger matrixof the materials prepared by the inventors' GF/PL process result, atleast in part, from greater uniformity of polymer distribution in thematerials and/or greater uniformity in size and distribution of porosityin the materials. SC/PL prepared polymers will not have such a uniformpore structure because the solvent evaporates from the polymer in anon-uniform manner and thus the polymer concentration changesnon-uniformly in the material. For instance, SC/PL materials typicallyhave non-uniformity because as the solvent evaporates the polymerconcentration increases at the bottom of the matrix, i.e. the area wherethe matrix touches the glass cover slip. In contrast, the GF/PLmaterials exhibit a very uniform pore structure indicating that thepolymer foams uniformly throughout the particulate bed during thegas-foaming step.

Alternatively, it is hypothesized that in the GF/PL process themechanical properties may be enhanced by tensile alignment of thepolymer chains may be occurring during the elongation which occursduring foaming. (Principles of Tissue Engineering, Academic Press, p.264 (1997).

In any event, it is of great advantage in tissue engineering and otherapplications that the materials of the invention can be prepared formaximizing of tensile strength and compression resistance since they canbe handled and manipulated without mechanical breakdown more readily andsurvive better in the environment in which they are used withoutmechanical breakdown. Further, the materials of the invention with bothtypes of porosity, preferably with interconnecting porosity, provide aunique and advantageous material for many applications. The process canprovide materials with a total porosity of, for example, from above 0 to97% or higher. Preferably, the total porosity ranges from 90-97%.

For the process, a mixture of polymer and particulate material is used.The mixture is preferably as uniform as possible and can be provided byconventional means. Preferably, the mixture is molded, for example bycompression molding at room temperature or other suitable temperature toeffect the molding, to the size and shape which is substantially thesame as that desired for its ultimate use.

The polymer and particulate materials should be selected so that theparticulate can be leached with a leaching agent which does not dissolvethe polymer or otherwise adversely impact on the material. Polymers andparticulates useful for the SC/PL processes discussed herein aregenerally useful for the GF/PL process of the invention. Further usefulmaterials are discussed below.

Any polymer into which gas can be dissolved and pores formed thereby andin which a particulate can be incorporated and leached therefrom can beused in the process. It is preferred, to facilitate dissolution of thegas, that the polymer be an amorphous or predominantly amorphouspolymer. However, if it is desired to use a crystalline polymer thecrystallinity can be reduced to a level such that the gas can bedissolved therein and then the crystallinity restored after formation ofthe pores. Depending upon the application of the materials, the polymermay be selected to be biodegradable or non-biodegradable. For manyapplications, such as tissue engineering, the polymer preferably isbiocompatible to the environment in which it is used. A preferred usefulclass of polymers are homopolymers and copolymers of lactic acid andglycolic acid, for example, poly-L-lactic acid (PLLA), poly-D,L-lacticacid (PDLLA), polyglycolic acid (PGA) and copolymers of D,L-lactide andglycolide (PLGA), particularly with 50% or more of the lactide in thecopolymer. Although under many conditions copolymers are preferred overhomopolymers, homopolymers may be preferred in some circumstances. Otheruseful polymers, for example, are aliphatic polyesters, such aspolyhydroxybutyrate, poly-ε-caprolactone. Further, polyanhydrides,polyphosphazines, polypeptides may be used.

Additionally, blends of different polymers may be used or polymers whichcontain other agents, particularly which effect the mechanicalproperties of the resulting matrix. For example, blends of differingPLGA polymers which have distinct properties can be used to takeadvantage of the properties of both. Also, other polymers can be blendedwith the PLGA polymers, particularly for modifying the mechanicalproperties thereof. For instance, a blend of a PLGA polymer and alginatematerial was found to provide a tougher matrix with greater elasticityand ability to withstand greater strain before breaking. Thus, it can beuseful, depending on the application, to blend polymers which result ina matrix with better pliability and/or strength. Blends with materialswhich act as plasticizers, toughening agents or modifiers of otherproperties are, therefore, useful in the invention. These materials caneither be polymers or smaller molecule agents which may act in atemporary manner and then diffuse from the matrix.

The polymer composition and molecular weight also have a large affect onthe three dimensional matrices' porosity and mechanical properties.Copolymers of PLGA were shown to foam to a much greater extent thaneither homopolymer PGA or PLLA. This finding is consistent with previousreports that the amorphous PLGA copolymers foam more than does thecrystalline polymer PGA (Mooney et al., Biomaterials, 17, 1417-1422,1996). This is likely due to an increased gas dissolution in amorphouspolymers as compared to crystalline polymers (D. F. Baldwin et al.,J.Eng. Mat. Tech., 117, 62, 1995; and D. W. Van Krevelen, Properties ofPolymers, Elsevier Publ., 1976). The molecular weight of the polymer hasa large effect on scaffold porosity. A polymer with a high molecularweight (large i.v.) did not form scaffolds with as high of porosity asthe same polymer with a lower molecular weight. The longer polymerchains of the high molecular weight polymer likely entangle to a greaterextent, thus providing a stronger resistance to expansion than theshorter polymer chains.

In one preferred embodiment, maximal pore formation can be achieved byuse of a low molecular weight amorphous copolymer of lactide andglycolide. These matrices will likely have great utility in theregeneration of oral tissues. They may be used alone as a GTR matrix.They may also be utilized to deliver growth factors in a sustained,local manner to promote regeneration. In addition, they could be used totransplant cells directly to a site to promote tissue regeneration fromthese cells and interacting host cells.

The leachable particulate is any particulate material which can beleached from the polymer matrix with a leaching agent. Preferred aresalts soluble in an aqueous medium, preferably water. As salts, NaCl, Nacitrate, Na tartrate, and KCl are useful particulate materials. Otheruseful particulates leachable by dissolution include, for example,gelatin, collagen and alginate particulates. It is also possible to useparticulates which are leachable by organic solvents where the solventdoes not adversely effect the polymer, however, this is not preferredsince such would mitigate the advantage of lack of need for an organicsolvent and lack of residue in the product. As discussed above, the sizeof the particulate will affect the size of the pores formed uponleaching of the particulate. Although not limiting of the invention, itis preferred that the particulate has an average size of from 10 to 500microns. This size will correspond approximately to the size of thepores formed by the leaching thereof.

A gas is dissolved in the polymer of the, preferably molded, mixture ofpolymer and particulate by subjecting the mixture to a pressurizedatmosphere of a gas which is inert to the system and will dissolve inthe polymer under suitable conditions. Suitable gases and conditions areknown from other gas-foaming processes, such as discussed in theBiomaterials article above, and they can generally be used herein.Preferred examples of suitable gas include CO₂, air, nitrogen, helium,neon, krypton, argon, xenon or oxygen. Also volatile liquids whichprovide a gas at the gas foaming temperature may be used, e.g. water.However, other gases or volatile liquids which form gases known to beuseful as blowing agents may also be used. These include, for example,fluorinated, including perfluorinated, hydrocarbons. Preferred for theseare aliphatic or cycloaliphatic fluorinated hydrocarbons of up to 8carbon atoms such as trifluoromethane, difluoromethane, difluoroethane,tetrafluoroethane, heptafluoroethane, perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoropentane, perfluorohexane,perfluoroheptane, pefluorooctane, perfluorocyclopentane,perfluorocyclohexane, hexafluoropropane and heptafluoropropane. Sulfurhexafluoride may also be a useful blowing agent. Other known blowingagents include alkanes such as propane, butanes and pentanes;cycloalkanes and cycloalkenes such as cyclobutane, cyclopentene andcyclohexene; dialkyl ethers such as dimethyl ether, methyl ethyl etherand diethyl ether; cycloalkylene ethers such as furan; ketones such asacetone and methyl ethyl ketone; and carboxylates such as formic acid,acetic acid and propionic acid.

The pressure is selected to facilitate dissolution of gas into thepolymer and will, thus, depend upon the gas used, the polymer used andthe temperature. Pressures of from about 600 to 900 psi are generallyuseful for CO₂ and PLGA polymers, although not limiting on theinvention. For example, gases at super- or sub-critical conditions caneven be used. Furthermore, a volatile liquid which can be dissolved inthe polymer and forms a gas upon imposition of the thermodynamicinstability can be used. As an example, CO₂ can be dissolved in amixture of poly[D,L-lactic-co-glycolic acid] polymer and NaClparticulate at a pressure of about 800 psi applied for about 48 hours toallow saturation.

The specific gas used in foaming can be a critical variable inproduction of porous matrices. The choice of gas used in foaming has alarge effect on the final scaffold structure. CO₂ produced highly porousmatrices, whereas N₂ and He led to no measurable pore formation. Theseresults are consistent with a number of previous studies in which CO₂has been used to create porous polymer structures (Mooney et al.,Biomaterials, 17, 1417-1422, 1996). While the exact mechanism underlyingthis result is not known, the greater degree of foaming experienced withCO₂ as compared to both N₂ and He may be the result of a specificinteraction between CO₂ and the carbonyl groups of PLGA (Kazarian etal., J. Am. Chem. Soc, 118, 1729-1736, 1996).

The gas equilibration time and pressure release rate affected theporosity and stability of the matrices, although not as strongly as theother variables.

In order to initiate nucleation of the dissolved gas and growth of gaspores in the material, a thermodynamic instability is created. Thisphenomenon is described for example by Park, Baldwin and Suh, “Effect ofthe Pressure Drop Rate on Cell Nucleation in Continuous Processing ofMicrocellular Polymers,” Polymer Engineering and Science, 35, pp.432-440 (1995). Preferably, this is done by lowering the pressure of thegas atmosphere, for example, down to atmospheric pressure over a shorttime period. The time period being, for example, from a few seconds toabout 15 minutes. The gas phase separates from the polymer via porenucleation and growth of the pores occurs through diffusion of gas intoareas adjacent the nucleation sites. The pore growth in turn reduces thepolymer density. Other methods for creating the instability, such asraising the temperature, may be used, but, are not preferred due to easeof processing. The pore structure and pore size of the gas pores formedwill be a factor of, among others, the type of gas used, the amount ofgas which will depend upon temperature and initial and final pressure ofthe gas atmosphere applied, the solubility of the gas in the particularpolymer, the rate and type of pore nucleation and the diffusion rate ofthe gas through the polymer to the nuclei. These and other factors canbe adjusted to provide gas pores of a suitable size. Sufficient gasshould be dissolved to cause formation of a continuous polymer matrixwhen the polymer expands during gas pore growth.

As a result of the thermodynamic instability, pore nucleation and gaspore formation and expansion, the polymer containing the particulatematerial forms a continuous phase, i.e matrix, around the gas pores.

The particulate is leached from the polymer with a leaching agent.Useful as leaching agent is any agent which will leach, e.g., dissolveand remove, the particulate from the polymer. As discussed above, anaqueous-based leaching agent, particularly water, is preferred. Theleaching of the particulate from the polymer forms the type of porosity,other than that formed by the gas-foaming, which as discussed above canprovide for an interconnecting pore structure.

The following embodiment is provided as a representative, non-limiting,example of the invention.

Discs comprised of polymer (e.g., poly[D,L-lactic-co-glycolic acid]) andNaCl particles were compression molded at room temperature, andsubsequently allowed to equilibrate with high pressure CO₂ gas (800psi). Creation of a thermodynamic instability led to the nucleation andgrowth of gas pores in the polymer particles, and the formation of acontinuous polymer matrix. The NaCl particles were subsequently leachedto yield macropores, and a macropore structure. The overall porosity andlevel of pore connectivity was regulated by the ratio of polymer:saltparticles. Both the compressive modulus (159±130 kPa for SC/PL vs.289±25 kPa for GF/PL) and tensile modulus (334±52 kPa for SC/PL vs.1100±236 kPa for GF/PL) of matrices formed with this approach weresignificantly greater than those formed with a standard solventcasting/particulate leaching process. The potential of these matricesfor engineering new tissue was demonstrated by finding that smoothmuscle cells readily adhered and proliferated on these matrices, formingnew, high density tissues (3×10⁷ cells/ml) in culture. This novelprocess, a combination of high pressure gas foaming and particulateleaching techniques, allows one to fabricate matrices from biodegradablepolymers with a well controlled porosity and pore structure.

The materials prepared by the process of the invention exhibit a widerange of utilities. They may be applied to any use which requires aporous polymeric material, particularly with an open pore structure.Further, the materials are particularly applicable for uses whereinorganic solvent residue is not tolerable, e.g. in applications whereinbiocompatability is desired. For instance, the materials are useful asmatrices in which cells are compatible and grow to achieve theirintended function, such as in tissue replacement, eventually replacingthe matrix depending on its biodegradability. Furthermore, the materialscan be used to provide matrices already bound to cells which may then besurgically implanted into a body. Further, the materials can be used asa sustained release drug delivery system, as wound healing matrixmaterials, as matrices for in vitro cell culture studies or uses similarthereto. The stable structure of the materials of the invention provideideal cell culture conditions.

The materials of the invention prepared by the GF/PL process generallyfurther have applications similar to those of materials prepared by theSC/PL and phase separation techniques, for example, in a variety of celltransplantation applications, including for hepatocytes (D. J. Mooney,P. M. Kaufmann, K. Sano, K. M. McNamara, J. P. Vacanti, and R. Langer,“Transplantation of hepatocytes using porous biodegradable sponges,”Transplantation Proceedings, 26, 3425-3426 (1994); D. J. Mooney, S.Park, P. M. Kaufmann, K. Sano, K. McNamara, J. P. Vacanti, and R.Langer, “Biodegradable sponges for hepatocyte transplantation,” Journalof Biomedical Materials Research, 29, 959-965 (1995)), chondrocytes andosteoblasts. S. L. Ishaug, M. J. Yaszemski, R. Biciog, A. G. Mikos;“Osteoblast Function on Synthetic Biodegradable Polymers”, J. of Biomed.Mat. Res., 28, p. 1445-1453 (1994). However, the materials of theinvention have better mechanical properties and avoid the problem oforganic solvent residue that may damage transplanted or migrating cellsand nearby tissue and/or inactivate biologically active factors.

Smooth muscle cells readily adhere to the matrix material of theinvention and create three-dimensional tissues within these porousstructures; thus, they provide a suitable environment for cellproliferation. In vitro experiments indicate concentrated cell growtharound the periphery of the matrix. This is likely due to O₂ diffusionlimitations to the cells at the center of the matrix because of thethickness (3.4 mm) of the sponge.

In addition, these matrices have a better potential to incorporategrowth factors than those prepared using organic solvents. The potentialproblem with organic solvents is that residue remains in these polymersafter processing may damage the transplanted cells and nearby tissue.Further, exposure to organic solvents would inactivate many biologicallyactive factors. Currently, incorporation of growth factors withbiomaterials are done using microspheres. This method also uses organicsolvents during fabrication. This disadvantage can be eliminated withthe matrix materials of the invention because the growth factor can beincorporated directly into the polymer matrix to obtain a betterrelease.

One preferred manner of incorporating growth factors in a matrix fortissue engineering and/or cell proliferation is to provide a growthfactor contained within a polymeric structure in particle form, e.g. asbeads microspheres, or blended with another polymer or other molecules,before adding to the PLGA for foaming. The polymeric structure can beformed of another copolymer of PLGA which degrades at a different ratethan the PLGA utilized to form the bulk of the matrix or from adifferent polymer material, such as an alginate or modified alginatematerial. Such a system provides an additional level of control over therelease kinetics of molecules from the matrices, and additional controlover their bioactivity because the growth factors contained within thepolymeric structure can be designed to provide a controlled releaseeffect therefrom in addition to the release kinetics provided by thematrix. The release is this situation will likely be controlled byeither disassociation of the factor from the bead, release from thePLGA, or both. Thus, a high degree of control over release kinetics isprovided over a potentially wide range. Further, multiple factors can beincluded in a matrix (in multiple types of the described particlesand/or in polymer comprising the bulk of matrix) which will release atvarying times. This will be useful if we want a cascade of growth factorrelease, or waves of release of the same factor (e.g., for use inimmunizations). Incorporation of the growth factors into these particles(e.g., alginate beads) is also more suitable for maintaining thelong-term bioactivity of the factors than if they were immobilizeddirectly in the polymer comprising the bulk of foamed matrix.

Highly porous matrices, for example, from PLGA, with a combination ofgas foaming and particulate leaching can be prepared by the invention.The method avoids the use of organic solvents or high temperatures andyields materials with desirable pore structures. It is possible tocontrol the porosity and pore structure of these matrices by varying theparticulate polymer ratio and particulate particle size for example.These matrices exhibit enhanced mechanical properties, and can beutilized to form three-dimensional tissues. This novel fabricationmethod can be used, for example, as an approach for drug and/or growthfactor incorporation into polymers used as tissue engineering matrices.

Another useful application for the polymer matrices of the invention isfor guided tissue regeneration (GTR). This application is based on thepremise that progenitor cells responsible for tissue regeneration residein the underlying healthy tissue and can be induced to migrate into adefect and regenerate the lost tissue. A critical feature of matricesfor GTR is the transport of cells into the matrix, a property which isdictated by the pore size distribution and pore continuity, i.e.,interconnectivity. The matrix must allow the desired cells to invade thematrix while preventing access to other cell types.

The materials of the invention, particularly as a polymer sponge made ofpoly(lactic acid) PLA, poly(glycolic acid) (PGA), orpoly(lactic-co-glycolic acid) (PLGA), having an impermeable layer on oneside can provide this selective permeability feature. The impermeablelayer is composed of the same polymers but without the extent ofporosity, and a variety of methods can be used to couple the impermeablelayer to the polymeric sponge.

In a particular embodiment which is representative of this utility, thepolymeric sponge is created by grinding PLGA followed by sieving toobtain particles with a diameter between 108 and 250 microns. Thesepolymeric particles are mixed with sodium chloride and pressed intoshape with a die at a pressure of approximately 1500 psi. Thepolymer/salt solid is then foamed by placing the solid in a pressurebomb and exposing it to CO₂ at a pressure of 800 psi for 48 hoursfollowed by a relatively rapid reduction in pressure. This reduction inpressure produces thermodynamic instabilities in distribution of CO₂causing pore formation. The polymer/salt solid is then placed in waterfor 24 hours to leach out the salt. Note that the water is changedduring the leaching process. This process produces a polymer sponge thatis greater than 95% porous. The degradation rate of the sponge can bemodified by varying the composition of lactic and glycolic acid.

An impermeable layer can be created on one side of the sponge by one ofthe following techniques, preferably performed before gas foaming of thematerial. The sponge can be pressed into shape on a layer of PGA at atemperature greater than the melting temperature for PGA. The melted PGAwill be able to adhere to the sponge thus forming a thin layer. Thislayer is impermeable because the foaming process and the leachingprocess have a negligible effect on pure PGA. An impermeable layer ofPLGA can also be created on the sponge by pressing the sponge onto alayer of PLGA. Spraying a solution of PLA in chloroform onto one side ofthe sponge can also create an impermeable layer. Further, it is possibleto use the same polymer material and alter the amount of leachableparticulate in each section so that one section forms an open porestructure and one does not. Also, by using different polymers, materialswherein one section foams, and the impermeable layer section does not,can be provided. Although PLGA does foam following release of pressurefrom the bomb, an impermeable skin forms on the thin layer of PLGA whichremains intact during the leaching process. Alternatively, following thefoaming and leaching process, the polymeric sponge can be dipped ineither melted PGA or in a solution of PLGA in chloroform. Theseprocedures can be used to create a sponge which has a porosity ofgreater than 95% with an impermeable side.

Similar methods can be applied to analogous materials, as discussedabove, to provide other sponge materials according to the inventionuseful for GTR applications.

The PLGA matrices also can provide a suitable substrate for boneformation. A critical feature of a matrix for replacement of bonytissues is its ability to provide an appropriate environment for tissuedevelopment and matrix mineralization. The ability of the GF/PL matricesto allow cell adhesion and tissue formation was assessed in vitro byseeding and culturing MC3T3-E1 cells, an osteogenic cell line, on PLGAscaffolds with techniques previously optimized for other cell types (Kimet al., Biotech. Bioeng., 57, p. 46-54, 1998). Cells adhered to theGF/PL matrix, proliferated, and began secreting extracellular matrixproteins, and by 4 weeks in culture patches of mineralization could beobserved. A new tissue with large areas of mineralization was formed by6 weeks. There was no observed change in the size and shape of thematrices over this time period suggesting they had sufficient mechanicalproperties to control gross formation of engineered bone tissue.

A critical feature of the matrix for use in guided tissue regenerationis the ability of cells to migrate into the matrix. Preliminaryexperiments confirm cells readily migrated into and throughout thematrix in vitro. This was expected as previous studies with these typesof matrices demonstrated fibrovascular ingrowth in vivo at a rate of0.1-0.3 mm/day (Mooney et al., 1994, supra).

Another potential application of these sponge materials for GTR is forthe treatment of periodontal disease. Periodontal disease ischaracterized by the loss of attachment of the periodontal ligament tothe alveolar bone. The epithelial cells of the gingiva begin to growinto the site where the periodontal ligament was attached. A sponge ofthe matrix material according to the invention with an impermeable sidecould be used to prevent the downgrowth of epithelial cells whileallowing the appropriate cells to occupy the porous sponge therebyregenerating the periodontal ligament. Further guidance as to suchapplication is provided by Shea et al., Tissue Engineering: Fundamentalsand Concepts, Chapter III.6, “Biodegradable Polymer Matrices in DentalTissue Engineering.”

For other applications in which cells are seeded or otherwiseincorporated and grown within the inventive matrices, incorporation andgrowth of the cells can be facilitated in a manner known in the art.Examples of such methods are provided in U.S. Pat. Nos. 5,041,138;5,567,612; 5,696,175 and 5,709,854; all of which are incorporated hereinby reference.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The entire disclosure of all applications, patents and publications,cited above and below, and of U.S. Provisional Application No.60/042,198, filed Mar. 31, 1997, are hereby incorporated by reference.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

EXAMPLES EXAMPLE 1

Matrix Processing

Pellets of an 85:15 copolymer of D,L-lactide and glycolide (PLGA) waspurchased from Boehringer Ingelheim (Henley, Montvale, N.J., USA), andutilized to fabricate polymer matrices in all experiments. The intrinsicviscosity of the polymer was about 1.3-1.7. Polymer pellets were groundusing a Tekmar grinder (Bel-Art Products, Pequannock, N.J., USA), andsieved to obtain particles ranging from 106 to 250 μm. In certainexperiments the polymer particles were mixed with sodium chlorideparticles (Mallinckrodt, Paris, Ky., USA). The salt particles weresieved to yield a range of sizes, and the weight ratio of NaCl:PLGAmasses ranged from 0 to 50. In all cases, the total mass of PLGA andNaCl was held constant at 0.8 g. The mixtures of PLGA and NaCl wereloaded into a KBr die (1.35 cm in diameter; Aldrich Chemical Co.,Milwaukee, Wis., USA), and compressed at 1500 psi for 1 minute using aCarver Laboratory Press (Fred S. Carver, Inc., Menominee Falls, Wis.,USA) to yield solid discs (thickness=3.4 mm). The samples were thenexposed to high pressure CO₂ gas (800 psi) for 48 hours to saturate thepolymer with gas. A thermodynamic instability was then created bydecreasing the gas pressure to ambient pressure. This led to thenucleation and growth of CO₂ pores within the polymer matrices. The NaClparticles were subsequently removed from the matrices by leaching thematrices in ddH₂O for 48 hours. All processing steps were performed atambient temperature.

Porous sponges were also fabricated using a previously described solventcasting-particulate leaching technique. (A. G. Mikos, A. J. Thorsen, L.A. Czerwonka, Y. Bao, and R. Langer, “Preparation and characterizationof poly(L-lactic acid) foams,” Polymer, 35, 1068-1077 (1994).) In thisprocess, PLGA was dissolved in chloroform (Mallinckrodt; Paris, Ky.,USA) to yield a solution of 10% (w:v), and 0.12 ml of this solution wasloaded into Teflon cylinders (diameter 0.5 cm;, Cole Parmer) packed with0.4 g of sodium chloride particles sieved to a size between 250 and 500mm. Following solvent evaporation, polymer films with entrapped saltparticles (3 mm thick) were carefully removed from the molds. The saltwas removed by immersing films in distilled water for 48 hrs.

Characterization

The porosity of samples was initially determined by gross measurementsand weights after processing using the following equation:

Eqn. 1: porosity(%)=1-[(weight/volume)/(density of polymer)]×100

The samples were imaged using a scanning electron microscope (ISI-DS130, Topcon Technologies, Pleasanton, Calif., USA). The samples weregold coated using a Sputter Coater (Desk II, Denton Vacuum, Cherry Hill,N.J., USA), and the microscope was operated at 10 kV to image thesamples. Polaroid 55 film was used for the photomicrographs.

Compression and tensile testing were performed on an MTS Bionix 100(Sintech, Research Triangle Park, N.C., USA). Samples were cut into 1×1cm squares for compression testing. For tensile testing, the samples(1×1 cm) were attached to cardboard using epoxy glue. A 7 mm slot wascut into the center of the card board and the sample was centered, thenglued to standardize the gage length. Compression and tensile tests wereperformed with a constant strain rate (1 mm/min). The moduli weredetermined from the slopes in the elastic portion of the stress-straindiagram.

Thermogravimetric analysis was utilized to determine the amount of saltresidue that remained in the sponge after leaching. Matrices were heatedfrom 150° C. to 300° C. at a constant rate of 10° C./min, and theresidual mass was monitored.

Cell Studies

Smooth muscle cells (SMC) were used in all experiments. SMCs wereisolated and cultured using a modification of the techniques describedin Rothman et al. (A. Rothman, T. J. Kulik, M. B. Taubman, B. C. Berk,C. W. J. Smith and B. Nadal-Ginard, “Development and characterization ofa cloned rat pulmonary arterial smooth muscle cell line that maintainsdifferentiated properties through multiple subcultures, ” Circulation,86, 1977-1986 (1992).) In brief, the cells were isolated from aortas of300-350 g adult male Lewis rats (Charles River Laboratories, Wilmington,Mass., USA) using an enzymatic dissociation. After fat, adventitia, andconnective tissue surrounding the arteries were removed by bluntdissection, the SM tissue was cut into multiple small pieces and placedinto a spinner flask containing an enzymatic dissociation buffer at 37°C. This buffer contains 0.125 mg/mL elastase (Sigma Chemical Co., St.Louis, Mo., USA), 1.0 mg/mL collagenase (CLS type I, 204 units/mg,Worthington Biochemical Corp., Freehold, N.J., USA), 0.250 mg/mL soybeantrypsin inhibitor (type 1-S, Sigma), and 2.0 mg/mL crystallized bovineserum albumin (BSA, Gibco/Life Technologies, Gaithersburg, MD., USA).After 90 minutes of incubation, the suspension was filtered through a100 5 m Nitex filter (Tetko, Inc., Briarcliff Manor, N.Y.) andcentrifuged at 200 g for 5 minutes. The pellet was resuspended in Medium199 (Sigma) supplemented with 20% (v/v) fetal bovine serum (FBS, Gibco),2 mM L-glutamine (Gibco), and 50 units/mL penicillin-streptomycin(Gibco). The cells were cultured on tissue culture plastic in ahumidified 5% CO₂ atmosphere with the medium (Medium 199, 10%(v/v) fetalbovine serum, 50 units/mL penicillin-streptomycin) changed every otherday. Cells at passage 17 were used in these experiments.

The matrices were seeded with SMCs by placing a 40 mL cell suspensioncontaining 3.14×101 cells/mL on top of each matrix and allowing the cellsuspension to absorb into the matrix. Matrices were contained in tissueculture dishes and incubated at 37° C. for −36 hours. Next, the polymermatrices were cultured for two weeks and placed in a spinner flask (100mL, Bellco Glass, Inc., Vineland, N.J., USA) stirred at 40 RPM. Thenumber of cells in the matrices was determined by measuring the DNAcontent in enzyme-digested triplicate samples using Hoechst 33258 dyeand a fluorometer (Hoefer DyNA Quant 200, Pharmacia Biotech, Uppsala,Sweden) as previously described. For scanning electron microscopicexamination, samples were fixed in 1% glutaraldehyde and 0.1%formaldehyde for 30 minutes and 24 hours, respectively, dehydrated in agraded series of ethanol/water solutions, dried, and then sputter-coatedwith gold. A scanning electron microscope (ISI-DS 130, TopconTechnologies) was operated at 10 kV to image samples. Histologicalsections were prepared by fixing cell-polymer matrices (10% formalin),dehydrating, embedding, sectioning and staining with hematoxylin andeosin or VerhoefUs using standard techniques.

Integrity and Porosity/Pore Structure of Foamed Matrices

Photomicrographs showed that gas foaming, alone, of solid polymer discsled to the formation of highly porous matrices. However, these matriceshad a nonporous skin on the external surfaces and the pores were largelyclosed, as expected from previous studies. (D. J. Mooney, D. F. Baldwin,N. P. Suh, J. P. Vacanti, and R. Langer, “Novel approach to fabricateporous sponges of poly(D,L-lactic-coglycolic acid) without the use oforganic solvents,” Biomaterials, 17, 1417-1422 (1996).) In contrast,gas-foaming and subsequent leaching of discs containing a highpercentage (95%) of large (250<d<425 μm) NaCl particles, according tothe invention, led to the formation of highly porous, open pore matriceswith no evidence of an external, non-porous skin. The pore structureobserved in cross-sections of these matrices was similar to thatobserved in cross-sections of matrices formed with a SC/PL technique.However, the pore structure of matrices formed from the SC/PL process isoften not uniform throughout the matrix due to evaporation of theorganic solvent and subsequent increase in the polymer concentration ofthe remaining solution entrapped within the salt bed. For example, thesurface of these matrices that is adjacent to the glass coverslip duringprocessing is shown in photomicrographs to be typically less porous thanthe remainder of the matrix. In contrast, the pore structure of gasfoamed-particulate leached (GF/PL) matrices was uniform throughout thematrix and on the exterior surfaces. TGA analysis of matrices indicatedthat negligible amounts of NaCl remained after leaching. There was atrace of a white residue left in the dish. To confirm that the gasfoaming was responsible for the formation of stable matrices, controlsamples were compression molded, but not foamed. Leaching of the NaClfrom these matrices led to complete breakdown of the matrices.

The ratio of NaCl:PLGA and the size of NaCl particles in GF/PL matriceswere next varied to determine the range of porosity and pore structurethat could be obtained with this process (Table 1). The gross porosityof these matrices increased from 85.1%±2.3 to 96.5%±0.5 as the ratio ofNaCl:PLGA was similarly increased. At cnstant NaCl (95%), the increasein salt particle diameter had very little effect on the overallporosity. However, photomicrographs showed that as the salt diameter wasincreased, the pore size increased in parallel.

The stability of the matrices was next assessed using compressive andtensile mechanical tests. In general, the GF/PL matrices exhibitedimproved mechanical properties as compared to the SC/PL matrices (SeeFIG. 1). The average compression moduli were 159±130 kpa and 289±25 kPafor the SC/PL and GF/PL matrices, respectively. The average tensilemoduli were 334±52 kPa for the SC/PL matrices and 1100±236 kPa for theGF/PL matrices (Table II). This data represents a 80% increase incompression strength and a 300% increase in tensile strength.

Tissue Development on Synthetic Matrices

The ability of the GF/PL matrices to allow cell adhesion and tissueformation was next assessed in an in vitro study. Photomicrographes showthat SMCs adhered to the GF/PL matrix and covered the available surfacearea following seeding. A significant increase in cell number was shownafter 2 weeks in culture. The average cell density was 1.7×10⁷ cells/mLand 3.05±10⁷ cells/mL at 0 and 2 weeks, respectively. This is a 43.8%increase in cell density. The cells filled the pores of the matrix andcreated a new three-dimensional tissue within the synthetic matrix.However, most of the cell growth occurred around the periphery of thematrix in a relatively uniform manner, and a low cell concentration wasobserved in the center of the matrices at 2 weeks. There was no observedchange in the size and shape of the matrices over this time period.

TABLE I Gross porosity of sponges. NaCl Concentration Diameter (μm) (%)106-250 250-425 >425 80 — 85.1 ± 2.3 — 90 87.3 ± 1.9 91.5 ± 1.4 — 9593.9 ± 0.9 94.6 ± 0.9 95.0 ± 0.8 97 — 96.5 ± 0.5 —

TABLE I Gross porosity of sponges. NaCl Concentration Diameter (μm) (%)106-250 250-425 >425 80 — 85.1 ± 2.3 — 90 87.3 ± 1.9 91.5 ± 1.4 — 9593.9 ± 0.9 94.6 ± 0.9 95.0 ± 0.8 97 — 96.5 ± 0.5 —

EXAMPLE 2 Growth Factor Release from Foamed Matrix Method

125I-labelled vascular endothelial growth factor (VEGF) was first addedto a solution of 1% sodium alginate, and then beads of this solutionwere gelled by injecting droplets into a aqueous solution containingcalcium chloride. The alginate beads (approximately 3 mm in diameter)were collected, rinsed, and lyophilized. The lyophilized beads weremixed with 85:15 PLGA and NaCl particles and the mixture compressionmolded and processed with the gas foaming/particulate leaching processas previously described. Following salt leaching and drying, thematrices were placed in serum free tissue culture medium and maintainedat 37° C. Medium samples were taken periodically, and analyzed for thecontent of 125I-VEGF (released from PLGA matrices). The released growthfactor was normalized to the total incorporated growth factor.

Results

An initial burst of approximately 20% of the incorporated growth factorwas noted in the first day, and a sustained release of growth factor wasnoted for the remaining 20 days of the experiment (See FIG. 2).

EXAMPLE 3 Growth Factor Delivery

One factor which may facilitate the development of tissues on thematrices is the delivery of growth factors into the local environment.The incorporation and release of growth factors from these matrices wasassessed in vitro using 125I-labeled vascular endothelial growth factor(VEGF). A substantial fraction of the drug was released during theparticulate leaching process; however, the remaining drug was releasedin a sustained manner during the 21 days of the experiment (FIG. 4).

EXAMPLE 4

Matrix Fabrication

Pellets of poly L-lactic acid [PLLA], a 50:50 copolymer of D,L-lactideand glycolide (50:50 PLGA) with intrinsic viscosity (i.v. of 0.2 dL/g),a 75:25 PLGA copolymer (i.v.=1.3), and an 85:15 PLGA copolymer(i.v.=1.4) were obtained from Boehringer Ingelheim (Henley, Montvale,N.J., USA). PGA, 50:50 PLGA (i.v.=0.8) and 85:15 PLGA (iv=0.63) werepurchased from Medisorb (Cincinnati, Ohio, USA). 85:15 PLGA (i.v.=3.63)was obtained from Purasorb (Lincolnshire, ill., USA).

The solid polymer (PLLA, PLGA, PGA) was ground (after freezing withliquid nitrogen) using a Scienceware Micro-Mill (Bel-Art Products,Pequannock, N.J., USA) and sieved to a diameter of 106-250 5 m. NaCl,obtained from Fisher Scientific (Pittsburgh, Pa., USA), was sieved to adiameter of 250-425 5 m for use in certain experiments. Solid polymerdisks were formed by placing 150 mg polymer (PGA, 50:50 PLGA, 75:25PLGA, 85:15 PLGA, and PLLA) into a round stainless steel KBr die withdiameter 1.35 cm (Aldrich Chemical Co., Milwaukee, Wis., USA) andcompressing for 60 seconds at 1500 psi in a Carver Laboratory Press(Fred S. Carver, Inc., Menominee Falls, Wis., USA). This method yieldssolid disks to be foamed. All samples were fabricated in triplicate.

The disks were foamed in a high pressure vessel using CO₂, N₂, or He at850 psi. After the disks were equilibrated (148 hours) with the gas, thepressure was reduced to ambient. The resulting thermodynamic instabilitycaused nucleation and growth of gas pores within the polymer matrix.85:15 solid polymer disks (i.v.=1.4) were foamed for 1 hour in CO₂ andthe pressure was released at different rates (1, 2.5, 5, 10 minutes) todetermine if the rate of pressure release affects the final structure ofthe sponges. All processing steps were performed at ambient temperature.

Polymer/NaCl disks were fabricated in a similar way using 40 mg polymerand 760 mg NaCl, compressed into disks. Following foaming, the diskswere placed in distilled water in order to remove the NaCl. Thisleaching solution was changed several times over the course of about 18hours. The disks were considered to be completely leeched when theleeching solution did not give a precipitate with AgNO3. If Cl− ispresent in solution, it precipitates with Ag+ to form a whiteprecipitate. The failure of this precipitate to form indicated that theNaCl is completely removed from the scaffolds. The disks were then airdried overnight, measured and weighed, and stored in a dessicator undervacuum. The polymer disks were measured and weighed immediatelyfollowing foaming, then stored in a dessicator under vacuum.

Characterization

In order to calculate the porosity of the foamed disks, a boley gaugewas used to measure the diameter and thickness of each disk. The diskswere weighed on a Mettler balance and the following equation was used:(d=polymer density, g=disk wt, cm3=calculated disk volume).

porosity=100 [1-(g/cm3)/d]

Several of the samples were imaged using a scanning electron microscope(ISI-DS 130, Topcon Technologies, Pleasanton, Calif., USA). The sampleswere gold coated using a Sputter Coater (Desk II, Denton Vacuum, CherryHill, N.J., USA) and the microscope was operated at 10 kV to image thesamples. Polaroid 55 film was used for the photomicrographs.

Compression testing was performed on an MTS Bionix 100 (Sintech,Research Triangle Park, N.C., USA). Only polymer/NaCl disks were used incompression tests because the solid polymer disks foamed to irregularshapes. A constant strain rate of 1 mm/min was used, and moduli weredetermined from the stress-strain curves.

Results

Foaming Solid Polymer Disks

In the first series of experiments, solid polymer disks were foamed toinvestigate the role of the gas type, pressure release rate, and polymercomposition and molecular weight on the porosity of polymer matrices.85:15 PLGA matrices were foamed for 1 hour with several different gases(CO₂, N₂, He). Significant porosity resulted from foaming with CO₂ ascompared to N₂ and He. The “prefoam” porosity refers to the calculatedporosity following disk preparation, but prior to high pressureequilibration (FIG. 4). Visualization of matrices foamed with CO₂revealed a highly porous matrix consisting largely of closed pores.

In the next study, the rate of release of pressure was varied from 1 to10 minutes total time. The porosity of the matrices was relativelyconstant regardless of pressure release rate, except in the case of avery rapid release, when the gas froze within the chamber. This led to asmall decrease in the matrix porosity (FIG. 2).

The effect of the polymer composition was investigated by usingdifferent copolymer ratios of PLGA (pure PGA, 50:50, 75:25, 85:15 PLGAand pure PLLA). Neither PGA nor PLLA foamed appreciably. The copolymersall foamed to a porosity greater than 90% (FIG. 6). In fact, the 75:25copolymer foamed so extensively that it did not maintain its integrityin the pressure release/gas expansion phase and literally fell apart.Hence, no porosity value could be calculated for that sample.

In order to study the effect of polymer molecular weight on poreformation, disks of 85:15 PLGA with intrinsic viscosity (i.v.) rangingfrom 0.63 to 3.59 dL/g were foamed in 850 psi CO2 for 24 hours with apressure release of 2.5 minutes. The high i.v. PLGA led to matrices withrelatively low porosity, whereas the lower i.v. PLGA resulted in muchhigher porosity (FIG. 7).

Foaming Polymer/NaCl Disks

In the second series of experiments, NaCl was incorporated into thepolymer disk for the purpose of creating an open pore structure.Different variables (equilibration time and polymer composition) werestudied in order to determine their effects on the structure andstability of the scaffolds. The results of the first series ofexperiments led us to use CO₂ as the foaming gas, and a pressure releasetime of 2.5 minutes in this series of experiments. Examination of atypical matrix formed by foaming 85:15 PLGA with NaCl in CO₂ shows ahighly porous structure with largely open, interconnected pores.

In the first study, the equilibration time was varied from 1 to 48hours. The porosity of the matrices was relatively constant forequilibration times greater than 6 hours, but decreased forequilibration times under 6 hours (FIG. 8a). Matrices fabricated withvarious equilibration times were subsequently tested to determine if theequilibration time affected their mechanical properties. Even thoughmaximal porosity was achieved with 6 hours of gas equilibration, astronger scaffold was produced with longer equilibration times (FIG.8b).

The polymer Composition was next varied to determine if results similarto those in the first series of experiments would be obtained.Copolymers of PLGA led to a much greater porosity than did thehomopolymers PGA and PLLA (FIG. 9a). Both the PLLA and PGA disksdisintegrated in the leaching process, indicating that little, if any,foaming had occurred. Even though all PLGA copolymers led to matriceswith similar porosities, the matrices fabricated from PLGA with higherlactic acid content were more rigid (FIG. 9b).

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanyingdrawings, in which like reference characters designate the same orsimilar parts throughout the several views.

FIG. 1: A graph comparing mechanical properties (tensile strength) ofSC/PL and GF/PL matrices.

FIG. 2: Shows the release profile of radiolabeled growth factor from thepolymer matrix according to Example 2.

FIG. 3: Shows the cumulative VEGF release over time for the matrixaccording to Example 3.

FIG. 4: The effect of gas type on porosity of matrices. 85:15 PLGA(i.v.=1.4 dL/g) disks were equilibrated for 1 hour in 850 psi gas priorto pressure release. The time for pressure release was 2.5 minutes.

FIG. 5: The effect of pressure release rate on porosity of PLGAmatrices. 85:15 PLGA (i.v.=1.4) disks were foamed for 1 hour in CO₂,with a pressure release time of 1 to 10 minutes.

FIG. 6: Porosity of matrices fabricated from different polymers.Polymers were exposed to 850 psi CO₂ for 24 hours with pressure releaseof 2.5 minutes.

FIG. 7: The effect of molecular weight on porosity of PLGA matrices.Matrices of 85:15 PLGA with varied intrinsic viscosity were foamed for24 hours in 850 psi CO₂ with a pressure release time of 2.5 minutes.

FIG. 8a: Porosity of matrices with varied equilibration times. 85:15PLGA (i.v.=1.4) and NaCl disks were foamed in 850 psi CO₂ for timeranging from 1-48 hours. The pressure release time was 2.5 minutes.

FIG. 8b: The elastic modulus of polymer/NaCl scaffolds fabricated withdifferent equilibration times. 85:15 PLGA (i.v.=1.4)/NaCl disks werefoamed in 850 psi CO₂ for 1-12 hours with 2.5 minute pressure release.

FIG. 9a: The effect of polymer composition on porosity of polymer/NaClscaffolds. Different copolymers of PLGA, PGA, and PLLA with NaCl werefoamed for 24 hours in 850 psi CO₂ with a pressure release time of 2.5minutes.

FIG. 9b: The elastic modulus of matrices formed with different polymercompositions. Different copolymer ratios of PLGA with NaCl were foamedfor 24 hours in 850 psi CO₂ with 2.5 minute pressure release.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A process for preparing a porous polymer materialwhich comprises forming pores in a polymer material which contains aleachable particulate by foaming and subsequently leaching out theparticulate material to form additional porosity wherein the polymermaterial comprises a homopolymer of a lactic acid or glycolic acid, acopolymer of a lactic acid and glycolic acid, an aliphatic polyester, apolyanhydride, a polyphosphazine, a polypeptide, a blend of a copolymerof a lactic acid and glycolic acid with an alginate, or any combinationof the above materials.
 2. The process of claim 1, wherein formation ofthe pores by gas foaming is conducted by subjecting a mixture ofparticles of the polymer and particles of the particulate to an elevatedpressure atmosphere of an inert gas such that the gas dissolves into thepolymer and then creating a thermodynamic instability such thatnucleation and growth of gas pores occurs and the polymer containing theparticulate forms a continuous matrix.
 3. The process of claim 2,wherein the mixture of particles of the polymer and particles of theparticulate is compression molded into a selected size and shape beforeformation of the gas pores.
 4. The process of claim 2, wherein thethermodynamic instability is created by reduction of the pressureatmosphere.
 5. The process of claim 2, wherein the gas is CO₂.
 6. Theprocess of claim 1, wherein the polymer is a biocompatible polymer. 7.The process of claim 1, wherein the polymer is a biocompatible andbiodegradable polymer.
 8. The process of claim 1, wherein the polymer isa homopolymer or copolymer of lactic acid and/or glycolic acid.
 9. Theprocess of claim 1, wherein the polymer is PLGA.
 10. The process ofclaim 1, wherein the polymer is a blend of a homopolymer or copolymer oflactic acid and/or glycolic acid with another polymer.
 11. The processof claim 10, wherein the polymer is a blend of a homopolymer orcopolymer of lactic acid and/or glycolic acid with an alginate polymer.12. The process of claim 1, wherein the particulate is a water-solubleparticulate.
 13. The process of claim 1, wherein the particulate is asalt.
 14. The process of claim 1, wherein the particulate is NaCl. 15.The process of claim 1, wherein the size and amount of the particulateis selected such that an interconnected pore structure in the porouspolymer material is formed.
 16. The process of claim 13, wherein theamount of particulate is at least 50% by volume of the mixture ofparticles of the polymer and particles of the particulate.
 17. Theprocess of claim 13, wherein the average particle size of theparticulate is from 10 to 500 microns.
 18. A porous polymer comprising apolymer matrix containing pores formed by gas foaming and pores formedby leaching out of a particulate from the polymer wherein the polymermaterial comprises a homopolymer of a lactic acid or glycolic acid, acopolymer of a lactic acid and glycolic acid, an aliphatic polyester, apolyanhydride, a polyphosphazine, a polypeptide, a blend of a copolymerof a lactic acid and glycolic acid with an alginate, or any combinationof the above materials.
 19. The polymer of claim 18, wherein the polymermatrix is a biocompatible and biodegradable polymer.
 20. The polymer ofclaim 18, wherein the polymer matrix is a homopolymer or copolymer oflactic acid and/or glycolic acid.
 21. The polymer of claim 18, whereinthe polymer matrix is PLGA.
 22. The polymer of claim 18, wherein thepolymer has an interconnected pore structure.
 23. The polymer of claim18, wherein the combination of pores provides a uniform open porestructure.
 24. The polymer of claim 18, wherein the polymer exhibits atensile modulus of 850 kPa or higher.
 25. A method for drug deliverywhich comprises introducing a drug contained within a porous polymer ofclaim
 18. 26. A method for drug delivery according to claim 25, whereinthe drug is a growth factor contained within the polymeric structure ofa polymer bead which is contained within the porous polymer.
 27. Amethod for tissue engineering which comprises introducing as a matrixfor the tissue a porous polymer of claim
 18. 28. A method for celltransplantation comprising administering a combination of a porouspolymer of claim 18 and cells for transplantation.
 29. A method for cellculturing which comprises culturing cells in the pores of a porouspolymer of claim
 18. 30. A polymer material which comprises a section ofporous polymer comprising a polymer matrix containing pores formed bygas foaming and pores formed by leaching out of a particulate from thepolymer and a section of impermeable polymer integrally connectedwherein the polymer material comprises a homopolymer of a lactic acid orglycolic acid, a copolymer of a lactic acid and glycolic acid, analiphatic polyester, a polyanhydride, a polyphosphazine, a polypeptide,a blend of a copolymer of a lactic acid and glycolic acid with analginate, or any combination of the above materials.
 31. The polymermaterial of claim 30, wherein the porous polymer has a uniform open porestructure and the impermeable polymer is of the same polymer materialbut without an open pore structure.
 32. A method for guided tissueregeneration which comprises introducing to the location requiringtissue regeneration a polymer material according to claim
 30. 33. Thepolymer material of claim 30, wherein the porous polymer and theimpermeable polymer are of different polymer material.
 34. The polymerof claim 18, which further comprises a drug contained within the poresof the porous polymer.
 35. The polymer of claim 18, which furthercomprises viable cells within the pores of the porous polymer.
 36. Thepolymer material of claim 28, which further comprises within the poresof the section of porous polymer viable cells for tissue regeneration.